Natural products play important roles in drug development. Ribosomally synthesized and post-translationally modified peptides (RiPPs) present a broad structural diversity, typically restricting conformational flexibility to allow better target recognition and to increase chemical, physical and proteolytic stability augmenting chemical functionality. For example, lasso peptides are a structural class of RiPPs exhibiting enzyme inhibitory, receptor antagonistic, antimicrobial or antiviral properties. Lasso peptides have unique mechanically interlocked topology in which the C-terminal tail is threaded through and trapped within an N-terminal macrolactam ring generated by an isopeptidic bond between the amino group of a glycine, alanine, serine or cysteine residue at position 1 and the side-chain carboxyl group of a glutamate or aspartate residue at position 7, 8 or 9 (FIG. 1). This threaded fold is maintained in the C-terminal tail region by steric hindrance, mediated by bulky side-chains above and below the ring, named plugs, and/or by disulfide bonds leading to a compact rotaxane type structure. As a consequence of their compact and interlocked structures, lasso peptides often display strong resistance against chemical and proteolytic degradation as well as, in many cases, thermally-induced unthreading.
More than 40 lasso peptides are now found in actinobacteria, proteobacteria, and firmicutes. Their size ranges from 13 to 24 residues, and are divided into three classes depending on the absence (class II) or presence of one (class III) or two (class I) disulfide bonds that can further stabilize the lasso structure (FIG. 1). Many lasso peptides were discovered through genome mining approach. The extraordinary mechanically interlocked topology of lasso peptides, together with their panel of biological activities makes them a promising scaffold for next generation drug design. These structures have not been reproduced using synthetic methods. However, unthreading of the C-terminal tail has been reported for several lasso peptides, yielding their corresponding (mostly) biologically inactive branched-cyclic topoisomers. The discovery and search for new lasso peptides as potential drug candidates requires high throughput analytical tools capable of differentiating them from their unthreaded branched-cyclic topoisomers.
Metal ions play a vital role in many biological processes. They act in a variety of important functions in protein systems including enzyme catalysis, protein folding, assembly, structural stability and conformational change. The presence or absence of a specific metal ion is crucial to the conformational space and/or chemical functionality of over one third of all proteins. Several structural studies aimed to determine the binding site locations, the nature of metal ion coordination, and the role of metal-ligand interactions on structure and function. Interactions between metal ions and biomolecules have been investigated using well established techniques such as circular dichroism, nuclear magnetic resonance (NMR) spectroscopy, and x-ray crystallography. Furthermore, the structures of metal-containing peptides and proteins in the gas-phase have been reported using mass spectrometry equipped with soft ionization sources such as electrospray ionization (ESI). Interestingly, the addition of metal ions often influences the fragmentation patterns upon activation. In addition, the potential of metal ions to differentiate isomer species, which present the same fragmentation pattern, has been shown using tandem mass spectrometry. All these results suggest the existence of highly-specific metal-binding sites. The interaction of peptides and proteins with metal ions has been a subject of considerable interest in ion mobility spectrometry coupled to mass spectrometry (IMS-MS) since the pioneering work of Clemmer and Jarrold. These studies showed the potential of IMS-MS to provide a more detailed understanding of the basic interactions that occur and represent an important step in the conformational engineering of peptides and proteins. Research on metal-peptide systems provides a basis for understanding metal interactions occurring in biochemically relevant systems.
Nearly all biomolecules have one or more chiral centers (typically on the C atoms) with geometries often crucial to their function. In enantiomeric pairs, all symmetry centers are inverted to form the mirror images. In particular, all α-amino acids (aa) except glycine have four different groups attached to the α-carbon, which allows left-handed (L) and right-handed (D) forms. From these, one can assemble peptides of any length comprising just L- or D-enantiomers and both forms (diastereomers or epimers). A species can have only one enantiomer, but numerous diastereomers with single or multiple D/L-substitutions in different positions.
No natural D-aa containing peptides (DAACPs) or proteins were known (except in bacterial walls) until the discovery of dermorphin in frog skin. About forty DAACPs are now found in eukaryotes such as arthropod, molluscan, and vertebrate. These peptides range from four to over fifty residues, and are epimers of all-L peptides with D-aa often located in the 2nd position from the N-terminus.
Such species have different conformations, resulting in distinct interactions with both chiral and non-chiral partners. Hence, DAACPs bind to receptors with different selectivity and affinity than the L-analogs, dramatically altering the biological function. For example, NdWFa enhances the heartbeat of sea slugs at 10−10 M whereas the L-analog NWFa is inactive even at 10−6 M. Many DAACPs were discovered by comparing the biological activities of natural and synthetic L-analog peptides. The unnatural stereochemistry of D-residues commonly renders DAACPs resistant to proteolytic degradation, making them a promising scaffold for next-generation drug design. Fundamentally, understanding the prevalence, substitution patterns, synthesis pathways, properties, and biomedical roles of DAACPs may help grasping the origin of extraordinary preference for L-aa across biology that is likely central to the genesis of life on Earth.
The number, abundance, and biomedical importance of DAACPs may be profoundly underestimated because of the paucity of analytical techniques for their detection and characterization. Unlike most post-translational modifications (PTMs), D/L-substitutions cause no mass shift for peptides or their fragments. Therefore, the standard mass spectrometry (MS) approach to PTM analysis (finding the precursors with mass shifts and tracking those for dissociation products to locate the PTM site) would not work for DAACP detection.
As the tools for identification and quantification of mature proteins, proteomics is focused on the characterization of proteoforms and revealing the activity-modulating impacts of distinct patterns of post-translational modifications (PTMs). Many proteoforms feature different number or type of PTMs, detectable by MS based on the mass increment. Others are isomers with identical PTMs on different amino acid residues. Such “localization variants” are individually distinguishable by unique fragments in tandem MS, particularly employing electron transfer dissociation (ETD) that severs the protein backbone while retaining weaker PTM links. The conundrum is that multiple variants frequently coexist in cells, but MS/MS cannot disentangle mixtures of more than two as those with PTMs on internal sites yield no unique fragments. This calls for separation of protein (and peptide) variants at least to binary mixtures before the MS/MS step. Dedicated liquid chromatography (LC) methods could resolve some variants for peptides in the “bottom-up” mass range (<2.5 kDa) usual for tryptic digests, but not larger “middle-down” peptides (2.5-10 kDa) or intact proteins. Splitting proteins into peptides using sequence-specific proteases precludes global PTM mapping by obliterating the proteoform-specific connectivity information between the modified peptides.
This problem is most prominent for histone proteins that combine exceptional importance to life with great diversity of PTM types and sites. Histones (H2A, H2B, H3, and H4) consisting of ˜100-140 residues are nucleosome core particles—the spools that store the DNA in cell nuclei and apparently regulate chromatin structure and function through dynamic reversible PTMs including methylation (me), dimethylation (me2), trimethylation (me3), acetylation (ac), phosphorylation (p), and others. Permuting their order and modulating the site occupation levels in this “histone code” may drastically alter the activity of whole genome, defined chromatin domains, genomic regions, and/or individual genes. Nearly all PTMs in histones are on the enzymatically cleavable N-terminal domains (“tails”) protruding from the nucleosome. The H3 tail of ˜50 residues (˜5.5 kDa) is cleavable by the endoproteinase Glu-C, and its characterization approaches that of intact histone.
Given the presence of natural isomers, such as, topoisomers, epimers, and proteoforms of biomolecules and their importance in biological processes, methods of identifying and purifying such isomers are desired.